Details - Shea Research Lab

technology feature
Close contact
306
Getting more physical
306
Sticking it to them
307
Journeys across the membrane
Nathan Blow
The technology used to deliver nucleic acids
into cells has undergone a renaissance in
recent years. Some scientists think it may
have spawned out of a growing interest in
studying difficult-to-transfect primary cell
types, whereas others suspect the increasing
desire of scientists to use high-throughput
screening approaches in their research is
leading developers to hunt for new ways
to deliver DNA and RNA faster and more
efficiently. Whatever the reason, novel
approaches along with improvements to
existing protocols for getting molecules
into cells, both in vitro and in vivo, are being
reported with greater frequency.
Here we take a look at four recent developments in the field of nonviral nucleic
acid delivery technology and explore how
these advances are affecting research in
areas ranging from stem cell differentiation
to drug development.
Finding the right vehicle
Scientists have developed both physical and
chemical methods for nucleic acid delivery.
Physical methods, such as electroporation
or particle bombardment, involve rapidly
changing the cell membrane to allow uptake
of DNA or RNA, and chemical approaches
often require attachment of the nucleic acids
to delivery vehicles, thereby creating complexes that can be shuttled into a cell.
According to Anne-Laure BolcatoBellemin, in vivo research manager at
Poly plus-transfection, for chemical
approaches the choice of delivery vehicle
will often be determined by its cargo. “For us,
polymers are more dedicated to gene delivery,
while lipids are being used for [small interfering RNA] delivery,” she explains.
Polymer-based vehicles, such as polyethylenimine (PEI), polyamine and cyclodextran,
Polyplus-transfection
© 2009 Nature America, Inc. All rights reserved.
From high-throughput electroporation platforms capable of transfecting thousands of different cells in a
day, to nanowires that puncture and deliver DNA to just a single cell, new technology is emerging to help
researchers with their changing gene delivery needs.
Diagram of the proposed mechanism for cellular
uptake of a cationic delivery vehicle carrying
siRNA for gene silencing.
act by binding and condensing DNA or RNA
into stable particles that can be taken up by
cells through endocytosis. Bolcato-Bellemin
says that researchers at Polyplus have developed a suite of PEI-based vehicles for in vitro
DNA delivery as well as labeled vehicles that
can be used to track PEI-DNA complexes
within a cell. Other developers including
Qiagen with their SuperFect and PolyFect
transfection reagents, and Fermentas with the
ExGen 500 reagentalso provide polymerbased solutions for in vitro cell transfection.
Lipid-based vehicles, which can be conjugated to or surround nucleic acids or other
molecules to be taken up by cells, have been
used for transfection for more than 20 years
and are among the most popular in vitro
delivery vehicles. Several well-established
lipid-based in vitro DNA transfection
reagents, such as InvivoGen’s LipoGen
and Invitrogen’s Lipofectamine 2000 and
Optifectas well as their first-generation
Lipofectin and Lipofectamineare currently available to researchers. But lipids are
also finding more and more use as vehicles
for the delivery of small interfering RNA
(siRNA) into cultured cells. Ambion (now
part of Life Technologies Corporation) has
developed a lipid-based siRNA delivery
reagent called siPORT NeoFX, Invitrogen
has modified their cationic lipid formulation in Lipofectamine for siRNA, Mirus
Bio has developed a combination cationic
lipid and polymer–based siRNA transfection reagent called TransIT-TKO and
Polyplus-transfection has made available the
INTERFERin siRNA transfection reagent.
Transfection of cultured cells is not the only
area for nucleic acid vehicle development as
some scientists have turned their attention
from cultured cells to in vivo delivery. Several
biotechnology and pharmaceutical companies, including Polyplus-transfection, Altogen
Biosystems, Alnylam Pharmaceuticals and
Calando Pharmaceuticals, are now using
cationic lipids, biodegradable polymers
and liposomes as vehicles to guide pieces of
DNA or siRNA around the human body for
delivery to specific cell types. Several of these
vehicles are now undergoing clinical trials
and new vehicle development continues to be
a rapidly growing field, with new carrier molecules on the horizon. Robert Langer’s group
at the Massachusetts Institute of Technology
recently reported the construction of a combinatorial library of lipid-like materials that
they call lipidoids to be potentially used in
siRNA delivery1. Still, both polymer and lipid
vehicles have drawbacks when it comes to
in vivo delivery. Whereas lipids are efficient
nature methods | VOL.6 NO.4 | APRIL 2009 | 305
Cellectricon
© 2009 Nature America, Inc. All rights reserved.
technology feature
The Cellaxess HT system was designed for high-throughput transfection to enable more effective
RNAi screening.
delivery vehicles with effects that tend to last
longer than polymers, according to BolcatoBellemin, lipids also tend to induce more
immune response than the polymers, which
is problematic for therapeutic purposes.
These drawbacks have led to the emergence of nanoparticle vehicles in recent
years as an alternative to potentially circumvent some delivery issues. But at the outset,
the biggest benefits of using nanoparticle
vehicles for in vivo gene delivery might be
in what researchers can see by using these
little particles. It turns out that the iron
oxide core within some newly developed
nanoparticles can be used to image DNA or
siRNA delivery within the body using magnetic resonance imaging technology, giving
researchers a much clearer understanding
of delivery efficiency and targeting.
Some developers have entered the world
of nanoparticle-based gene delivery. In
2007, Sigma Aldrich introduced the N-TER
Nanoparticle siRNA Transfection system,
which allows for the conjugation of an siRNA
to the surface of the nanoparticle through
the N-TER peptide for in vitro delivery
applications, and two other developers, OZ
Biosciences and nanoTherics, are now offering magnetic nanoparticles for the delivery of
nucleic acids to cells.
In the end, regardless of the vehicle,
Bolcato-Bellemin says that when it comes to
nonviral delivery of nucleic acids, the goal is
straightforward. “The most important thing
is to transfect the highest percentage of cells
306 | VOL.6 NO.4 | APRIL 2009 | nature methods
with the lowest amount of DNA or RNA possible [while still achieving the desired result],”
she says, adding that in this way problems
such as cell toxicity, off-target effects and
immune response can be greatly reduced or
even eliminated altogether.
Close contact
“I think of [our approach] in many ways as
mimicking what nature does,” says Lonnie
Shea, professor in the chemical and biological engineering department at Northwestern
University, when discussing his group’s work
on substrate-mediated gene delivery.
The idea behind Shea’s delivery method is
simple: if a DNA–delivery vehicle complex is
tethered to a substrate surface to which the
cells are adhering, the cells and delivery vector are effectively co-localized, which should
result in more efficient gene delivery. But his
major challenge over the past few years has
been to fine-tune the associations between
the delivery complex and substrate.
Shea’s group initially opted for a biotinavidin interaction to tether polymer complexes of DNA to the substrate surface but
finding optimal combinations took time.
On one hand, he says: “If you have too much
biotin on the complex, then the binding is
too strong, and the cells cannot internalize
the DNA.” On the other hand, the use of too
little biotin results in mass mobilization, and
decreases efficiency and localized delivery.
Shea has also investigated the immobilization by nonspecific interactions between
the transfection reagent and the surface, and
their data suggest that effective gene delivery
results from displacement of the complexes
from the substrate by adsorbing proteins.
“If we immobilize complexes and wash with
phosphate-buffered saline the complexes are
stable and do not come off the surface. But
if you add serum proteins, then you see the
complexes being displaced to the surface.”
As displacement of complexes appears to
be necessary for uptake by cells, Shea says it
is important to have proper substrate surface composition. “If the associations are
too strong, then uptake is limited and the
complexes may lose their architecture, which
limits their efficacy,” he says.
Shea has transfected DNA in polymer
complexes, lipid complexes, as well as in
lentiviruses attached to the substrate surface. One of the benefits of such close contact between the cells and delivery vehicle
is that less DNA is needed for transfection,
enhancing delivery efficiency.
Shea’s group is now moving toward in vivo
applications using his substrates. “What the
substrate will do is retain the DNA for longer periods of time, preventing it from being
degraded,” he says, which could improve
gene delivery in clinical applications in which
complexes and viral vehicles are often rapidly
cleared from the body. “An advantage of this
approach is that the material can be processed by a variety of techniques, and then
the vectors can be immobilized, which stabilizes and increases the activity of the vector.
The material can then be implanted, and we
can look at the effects when those complexes
are delivered locally and stabilized against
nuclease activity.”
But as others have discovered with siRNA
delivery, delivery in the in vivo world is very
different from in vitro. “We have had great
success in vitro with efficient delivery to a
variety of cell types, but in vivo the environment around the cell is different, creating
new challenges,” notes Shea.
Getting more physical
For Johan Pihl, product manager at
Cellectricon, it took the advancement of
technology in another field to help him see
the next step for his company. “We believed
that we had this very potent method for gene
delivery, but at the time we did not know
quite what to do with it,” says Pihl.
That was five years ago. Then Pihl and his
colleagues started to see the RNA interference (RNAi) field “cook” with more and more
researchers interested in doing high-through-
© 2009 Nature America, Inc. All rights reserved.
P. Yang
Technology feature
Gene delivery can be accomplished by culturing cells on a bed of nanowires that have DNA on the
surface.
put screens along with core screening centers
springing up around the world, and he knew
their next step. “We realized this was where a
high-throughput transfection approach was
most needed since transfection is one of the
major bottlenecks in RNAi screens.”
So in 2006 developers at Cellectricon started working on plans to adapt their Cellaxess
gene delivery technologywhich is based
on electroporation where electric fields permeabilize cell membranes to deliver DNA or
RNAinto a high-throughput automated
instrument.
“We use a capillary tube equipped with
electrodes to deliver electric fields to the cells,”
explains Pihl. The idea for their new instrument was to take advantage of the small size
of these capillary tubes by bringing a series of
them into close proximity to adherent cells
located in individual wells on a microtiter
plate. An electrical field could then be applied
and delivered to each individual capillary to
transfect multiple cell populations.
Over the next two years engineers at
Cellectricon added the necessary automation components to their instrument. These
additions allowed for the transfer of buffers,
addition of siRNA to the cells before transfection, switching of liquid-handling tips with
electroporation units to electroporate cells
and finally the addition of cell culture media
for recovery after transfection. The system,
called Cellaxess HT, was introduced in April
2008 and is capable of transfecting 50,000
wells of cells per day.
Other companies have also developed
high-throughput solutions for gene delivery. The bioscience division of Lonza
now provides the 96-well microtiter plate
Nucleofector kit for transfection of stem
cells, which like the Cellaxess HT system
uses electroporation. Another company,
MaxCyte, has developed an electroporationbased approach in which cells in a flowing
stream are passed through an electroporation unit. Both the capillary electrode
approach taken by Cellectricon and the flow
design of MaxCyte enable lower currents
to be used than traditional electroporation
approaches, resulting in the generation of
less heat and improved cell viability after
transfection.
High-throughput transfection systems
could make RNAi screening faster and more
efficient in the future, but it is only one part
of the process. “Once you overcome the
transfection bottleneck, you still have the
problem of establishing a stable and good
assay that will give you hits at the end of
the day,” says Pihl. But he hopes that having
instruments dedicated to transfection will
give researchers more time to focus on their
downstream assays.
Sticking it to them
Peidong Yang, professor of chemistry at
the University of California, Berkeley,
thought his new delivery approach was a
long shot. “My intuition was that it would
kill the cell,” he recalls. And who could
blame him? The idea of puncturing a cell
with an electrical wire to deliver DNA does
seem a bit extreme at first.
The idea actually came from an informal
chat between Yang and his colleague Bruce
Conklin, a professor also at University of
California, San Francisco, a couple years ago.
Conklin was interested in using electrodes
nature methods | VOL.6 NO.4 | APRIL 2009 | 307
© 2009 Nature America, Inc. All rights reserved.
technology feature
to look at stem cell differentiation, while
Yang’s group had been working on nanowire-based nanoelectronics and probes.
“That was the starting point: nanowires as
electrodes for guiding stem cell differentiation,” says Yang.
But first things first, and for Yang and his
group this meant determining whether stem
cells could be punctured with nanowires
and actually survive. “What we saw was that
with sub-80-nanometer nanowires the cells
were ok,” says Yang. “At least in terms of not
changing the lifetime of the stem cell significantly.” But there was a strong size dependency in the cell’s ability to survive puncture
with the nanowire; wires over 200 nanometers killed cells.
Armed with the knowledge that cells can
in fact survive with unaltered lifetimes,
Yang fabricated an array of 80-nanometer
nanowires functionalized with DNA to
test whether the wires could also deliver
DNA. Although the cells survived the
puncture and could take up DNA, the
308 | VOL.6 NO.4 | APRIL 2009 | nature methods
results showed that these nanowires may
not be ready for most researchers’ gene
delivery needs yet. “I mean we are talking
about a low percent of transfected cells. It
is very inefficient,” notes Yang. He was not
surprised by the low efficiency because
the nanowires were made of silicon and
the DNA was physically absorbed to the
surfacenot the most efficient possible
attachment. Still, for his and Conklin’s
purposes the efficiency was more than
adequate, giving them the ability not only
to deliver small molecules on the nanowires but also electrically stimulate the
stem cells at the same time.
“Electrical stimulation of stem cells is
another way to think about how to program
the cell differentiation,” says Yang. Although
it is still very early in their research, and not
much is known about the effects of electrical stimulation on stem cells, Yang is
encouraged by the fact that they now have
a single platform to test both chemical and
electric stimulation.
And for those researchers and developers
interested in improving upon the current
efficiency of nanowires for gene delivery,
Yang thinks changing the surface of the wire
could be the way to go. “We are suggesting
that if anyone wants to improve the efficiency, one can think about using surface functionalization to covalently attach DNA and
maybe use photochemistry to cleave those
bonds inside the cell.”
While polymer, lipid and nanoparticle
delivery reagents along with electroporation techniques continue to be refined and
improved, the newer approaches, such as
nanowires and substrate mediated delivery,
demonstrate that there still is a lot to learn
when it comes to gene delivery.
1. Akinc, A. et al. A combinatorial library of lipid-like
materials for delivery of RNAi therapeutics.
Nat Biotechnol. 26, 561–569 (2008).
Nathan Blow is the technology editor
for Nature and Nature Methods
([email protected]).
technology feature
© 2009 Nature America, Inc. All rights reserved.
SUPPLIERS GUIDE: COMPANIES OFFERING GENE DELIVERY
PRODUCTS AND SOLUTIONS
Company
Web address
Ambion Inc
http://www.ambion.com/
Altogen Biosystems
http://www.altogen.com/
Applied Biosystems
http://www.appliedbiosystems.com/
Asuragen
http://www.asuragen.com
BD Biosciences
http://www.bdbiosciences.com/
Bio-Rad Laboratories Cenix
BioSciences
http://www.cenix-bioscience.com/
CombiMatrix
http://www.combimatrix.com
Dharmacon
http://www.dharmacon.com/
Eurogentec
http://www.eurogentec.com/
Exiqon
http://www.exiqon.com/
GeneTools
http://www.gene-tools.com
genoway
http://www.genoway.com/
Imgenex
http://www.imgenex.com/
Integrated DNA Technologies
http://www.idtdna.com/
Invitrogen
http://www.invitrogen.com/
Invivogen
http://www.invivogen.com/
Cellectricon
http://www.cellectricon.com/
Lonza/Amaxa biosystems
http://www.amaxa.com/
MaxCyte
http://www.maxcyte.com/
Mirus Bio
http://www.mirusbio.com/
MWG Biotech
http://www.mwg-biotech.com/
NanoLab Inc
http://www.nano-lab.com/
nanoTherics
http://www.nanotherics.com/
New England Biolabs
http://www.neb.com/
Ocean Nanotech
http://www.oceannanotech.com/
OligoEngine
http://www.oligoengine.com/
Thermo Fisher Open Biosystems
http://www.openbiosystems.com/
OZ Biosciences
http://www.ozbiosciences.com/
Tecan Group LTD
http://www.tecan.com/
Polyplus-transfections
http://www.polyplus-transfection.com/
Promega
http://www.promega.com/
Qiagen
http://www1.qiagen.com/
Sigma Aldrich
http://www.sigmaaldrich.com/
Stratagene
http://www.stratagene.com/
Sylentis
http://www.sylentis.com/
Targeting Systems
http://www.targetingsystems.com/
Vectalys
http://www.vectalys.com/
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